The present invention relates to retargetting of antibodies to a site or antigen for which they have no functional specificity under normal circumstances. A method is described employing an antigen-specific binding substance which possesses at least two specificities; one specificity for the target site, the other capable of binding to part of an antibody molecule. In this manner, antibodies with no specificity for the antigen target may be brought into proximity with the antigen via the antigen-specific binding substance. This principle is advantageous for re-targeting antibodies in the circulation to sites of disease within the body, e.g. tumours or sites of is viral, bacterial or parasitic infection or combinations thereof. This principle may also be applied to block inappropriate immune responses exemplified by autoimmune disease or hypersensitivity reactions. Retargetting can be achieved with conventional bispecific antibodies, e.g. prepared chemically or from hybrid hybridomas, or using the novel bispecific antibody fragments, diabodies (P. Holliger et al Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993 and PCT/GB93/02492).
Antibodies are proteins elaborated by B-lymphocytes to play a key role in the specific arm of the vertebrate immune system. This arises from their collective capacity to bind to an enormous diversity of antigen structures, with individual antibody molecules capable of precise specificity for their cognate antigen. The bulk of the antibody population is found in abundance in the blood and interstitial fluids, with minor types located at mucosal surfaces such as the intestinal lumen. An antibody binding to a foreign organism or a tumor cell marks it for destruction by the antibody encoded effector functions of the immune system. Destruction may be effected by either the complement cascase or antibody directed cell-mediated cytotoxicity (ADCC). ADCC is mediated through binding of antibody Fc regions to their Pc receptors on e.g. macrophages, eosinophils, K cells but also basophils and mast cells. Interaction with Fc receptors mediates not only cytolysis but also phagocytosis and immune clearance. Ig isotypes differ markedly in the spectrum of effector functions they recruit.
The immune system operates natural checks and balances to prevent production of antibodies with specificity for the host, so-called xe2x80x98self-antigensxe2x80x99. occasionally, the system breaks down causing autoimmune disease. Self-tolerance is one reason why the immune system may not destroy tumours and other malignancies, since these derive from host cells growing abnormally.
It has proved possible to use antibodies in medical intervention, using antibodies manufactured outside the body. Techniques for immortalisation of B-lymphocytes has enabled manufacture of monoclonal antibodies for a range of commercial applications in science and human health-care (Clinical Applications of Monoclonal Antibodies, E. S. Lennox, Ed. British Medical Bulletin 1984. Churchill-Livingstone). Moreover, an understanding of the genetic and physical structure of antibodies has enabled their manipulation outside of the immune system, through the use of molecular biology techniques, especially using phage display technology (WO 92/01047; WO 92/20791; WO 93/06213; WO 93/11236; WO 93/19172; WO 94/13804).
Structurally, the simplest antibody (IgG) comprises four polypeptide chains inter-connected by disulphide bonds. The light chains exist in two different forms called kappa (K) and lambda (X). Each chain has a constant region (C) and a variable region (V). Each chain is organised into a series of domains. The light chains have two domains, one corresponding to the C-region (CL) and the other to the V-region (VL). The heavy chains have four domains, one V-region domain (VH) and three C-region domains, CH1, CH2 and CH3. The basic IgG antibody is Y-shaped; the two arms (tip of the Y, each being an xe2x80x98Fabxe2x80x99 region) contain a VH and a VL domain associated with one another. It is this pair of V-regions that differ from one antibody to another (owing to amino acid sequence variations), and which together are responsible for recognising the antigen and providing an antigen binding site (ABS). In even more detail, each V-region (whether heavy chain or light chain) consists of three complementarity determining regions (CDRs) separated by four framework regions (FR) The CDR""s are the most variable part of the variable regions, and they perform the critical binding function. The CDR regions are derived from many potential germline sequences via a complex process involving recombination, mutation and selection.
It has been shown that the function of binding antigens can be performed by fragments of a whole antibody. Example binding fragments are (i) the Fab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fd fragment consisting of the VH and CH1 domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward, E. S. et al., Nature 341, 544-546 (1989)) which consists of a VH domain; (v) isolated CDR regions; (vi) F(abxe2x80x2)2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) bispecific single chain Fv dimers (PCT/US92/09965) and (viii) diabodies, bivalent or bispecific fragments constructed by gene fusion (P. Holliger et al, supra; WO 94/13804. Diabodies are discussed further infra. Bispecific fragments are especially well suited to the current invention.
Whereas the V-domains (and fragments containing V-domains) are largely responsible for interacting with antigen, the C-domains recruit effector functions. The type of effector function recruited is largely governed by the class of C-domain (the isotype; M. Bruggemann et al J. Exp. Med. 166 1351 1987; L. Riechmann et al Nature 332 323 1988; J. Greenwood et al Eur. J. Immunol. 23 1098-1104 1993). In this way, antibodies, which have evolved to combat pathogens, bind to antigens on the pathogen and in so doing initiate an appropriate immune response aimed at destroying the invader. For example, C-domains of the IgG1 (xcex31) isotype can kill cells by triggering the complement cascade at the cell surface, resulting in lysis, or through binding C-domain receptors (Fc receptors) on specialised phagocytic and killer cells through ADCC. On another hand, antibodies of the IgG4 isotype (xcex34) appear actively to block a response. In the context of the present application this blocking is considered to be an effector function which can be recruited to a chosen target. The binding sites for complement and Fc receptors map to the CH2 domain, sequence variation between CH2 domains of the different isotypes results in different strengths of interaction with complement and Fc receptors. All isotypes except IgE require that the C-domain is correctly glycosylated.
By association of the V-region with a given C-region isotype, an appropriate immune response can be triggered when the antibody binds to antigen. Because the type of immune response is governed by the isotype, artificially-made antibodies can be endowed with appropriate constant regions to be used therapeutically, for example to destroy tumour cells (Hale, G et al., Lancet ii, 1394-1399 (1988)).
If an antibody is to be used in such a way that requires recruitment of natural effector functions, then the antibody (except for the IgE isotype) must be manufactured in eukaryotic cells in order that the protein is glycosylated. Unfortunately, the type and extent of glycosylation varies with eukaryotic cell-type and culture conditions (Borys, M. C. et al., Biotechnology 11, 720-725 (1993)), and this can dramatically shorten their longevity in the circulation as well as adversely influencing recruitment of effector functions. There is the added risk that an inappropriately glycosylated antibody will be immunogenic, limiting the duration of the therapy.
One way of circumventing the need for correctly glycosylated constant regions is to manufacture antibodies comprising at least two different antigen-binding sites. These are known as bispecific antibodies and they can be manufactured in a variety of ways (Holliger, P. and Winter G. Current Opinion Biotechnol. 4, 446-449 (1993)). Again using tumour killing as an example, one antigen binding site is directed against a tumour marker whereas the other can be directed against an antigen present on an effector cell-type. Bispecific antibodies incorporating a specificity for the T-cell co-receptor CD3 have been shown to inhibit tumour growth (Titus, J. A. et al., J. Immunol. 138, 4018-4022 (1987)) and to cure lymphoma (Brissinck J. et al, J. Immunol. 174, 4019-4026 (1991)). In this way the interaction between Fc region and effector cell is replaced by direct interaction between one of the antigen binding sites and the effector. Diabodies directed against carcinoembryonic antigen (CEA; a human tumour cell marker) and CD16 (on human T lymphocytes) have been demonstrated to mediate lysis of human tumour cells on addition of peripheral blood lymphocytes (WO 94/13804).
The present applicants have realised that the direct interaction between the C-region of the antibody molecule and the effector results in limited activation of the immune system, and that it would be advantageous to activate (or indeed shut down) immune responses at a given target to a much greater degree. The applicants have further realised that such modulation may be achieved by redirecting naturally occurring antibodies to a site or target for which they do not necessarily possess specificity. The present applicants have realised in addition that this principle may be brought into effect through the use of binding substances which possess two or more specificities. One of many examples is a bispecific antibody which incorporates specificity for other antibodies. An antibody with specificity for a tumour cell and, for example, IgG1 constant regions will bind to the tumour in situ and accumulate IgG1 antibodies present in the circulation, such that IgG1-specific effector functions are called down at the tumour site. Antibodies in the serum of an individual are native to that person and therefore will be functional in activating complement or ADCC. The principle of indirect recruitment is beneficial over direct interaction with effector cells for several main reasons.
Firstly, there is evidence for the existence of antibody networks in the immune system, in which naturally occurring anti-antibody specificities build a branching mass of antibodies upon and around antibodies complexed at a target site (A. S. Perelson Immunol. Rev. 110 5-36 1989; antibody networks are reviewed in N.J. Calvanico Dermatol. Clin. 11 379-389 1993). It is is thought that this serves to amplify the effect of binding a few molecules of antibody to a target such that a small degree of specific binding can trigger a disproportionately large effector response. This contrasts with direct binding to effector cells or triggering of complement, since in this instance binding is stoichiometric (one antibody per antigen at most) rather than multiplicative.
A second reason why this arrangement is beneficial relates to control of serum half-life. Correctly glycosylated antibodies have fairly reliable serum clearance rates, the rate of turnover being different for different isotypes. For example IgG1 has a serum half life in the order of 21 days, whereas on the other hand, IgG3 and IgE are turned over in a matter of 1-2 days. The duration of the therapeutic effect may be controlled by the half-life of the administered bispecific antibody, e.g. diabody. The half-life is likely to depend on its binding affinity (and kinetics) for the targetted antibody and antigen and on the serum concentration of the antibody target.
Thirdly, this approach can be used in site-specific immunosuppression. Some antibodies, such as IgG4, actively prevent immune responses by blocking the epitopes. Indeed, some parasites are known exploit this property to escape immune attack (A. Capron et al Mem. Inst. Oswaldo Cruz 87 Suppl.5 1-9 1992), their antigens inducing antibody production of the correct specificity but with C-region isotypes incapable of inducing killing. This principle can be extended within the scope of the present invention to uses such as alleviation of autoimmune disorders such as rheumatoid arthritis and myasthenia gravis. In this case the bispecific antibody has specificity for the target epitope and for example, IgG4. However, patients may need to be screened for the ability of their immunoglobulin IgG4 to recruit effector functions, since the ability to do this appears to vary between individuals (Greenwood et al, supra).
Fourthly, in vivo, the individual""s natural allotypes are recruited so the need for matching the individual""s and the therapeutic antibodies allotypes is eliminated.
It will be clear to those skilled in the art that there exist many ways of putting this principle into operation. For example, naturally occurring or genetically engineered binding substances other than antibodies could be incorporated into a multiply-specific substance described herein. Examples include lectins, Fc-binding proteins such as-protein A or protein G, receptors such as Fc receptors and components from the complement system. Small molecules such as peptides, nucleic acids or naturally-occurring, partially synthetic or synthetic chemicals can also be used. The aforementioned can be used in any order, number and combination to create multiply-specific is substances described herein for use in therapy, diagnosis and scientific research. However, the use of antibody or a fragment thereof is preferred. Especially preferred are antibody fragments such as (Fab)2 and-diabodies lacking Fc regions, for reasons explained below. It should also be noted that, unless the context demands otherwise the term antibody is used herein (and commonly in the art) to include antibody fragments, both synthetic and naturally occurring, i.e. molecules comprising an immunoglobulin binding domain.
In the preferred embodiment, the multiply-specific substance described herein is a bispecific antibody capable of binding to an appropriate antibody isotype. xe2x80x9cDiabodiesxe2x80x9d may be particularly advantageous for the purpose since they can be readily constructed and expressed in E.coli. Diabodies of appropriate binding specificities can be readily selected using phage display (WO 94/13804) from libraries. If one arm of the diabody is to be kept constant, for instance, with a specificity directed against an immunoglobulin light chain, then a library can be made where the other arm is varied and an antibody of appropriate specificity selected.
Although any type of bispecific antibody molecule could be used for retargetting antibodies, it is preferable to use (Fab)2, scFv dimers or diabodies rather than whole antibodies. The presence of Fc in whole antibody may cause complications in vivo arising from direction to non-specific sites, especially to Fc receptors. Diabodies can be constructed without Fc, using only variable domains, avoiding this potential problem. In vitro, the simplicity of making bispecific diabodies, as opposed to bispecific whole antibodies, makes them the antibody form of choice.
One aspect of the present invention provides a method of recruiting an antibody mediated effector function to a target, the method employing a multi-specific binding substance having anti-antibody binding specificity and binding specificity for a target. This is illustrated in FIG. 1. Binding of the multi-specific binding substance to the target and to antibody allows recruitment of antibody-mediated effector function to the target. The binding substance is bound to antibody and to the target where it mediates the effector function of the antibody, generally, the effector function is the natural one of the bound antibody (e.g. ADCC, complement fixation or blocking, as discussed). The antibody may be any isotype, e.g. IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE, for recruitment of associated effector functions. Preferably the anti-antibody binding specificity of the binding substance is for the constant region of antibodies of one or more isotypes. Use of isotype-specific anti-antibody binding specificity enables choice of effector function recruited.
Human IgG1, IgG3 and IgM are particularly valuable for complement fixation and IgG1 and IgG3 are particularly valuable for ADCC. Then, the multi-specific binding substance will have binding specificity for a constant region of the isotype. IgM molecules are particularly useful in agglutination assays. IgG4 is the most suitable isotype for blocking antibodies, since it does not in general recruit antibody directed cellular cytotoxicity or complement. It may be valuable in some cases to use an isotype which does not activate complement to too great an extent, to prevent a toxic response. For recruitment of phagocytosis, IgG1 may be particularly suitable. Mast cells may be recruited via IgE antibodies. This may make them of value for cancer cell killing, but may limit their use for other applications (WO 92/11031).
Specificity directed against light chains allows recruitment of a spectrum of antibody isotypes including those which activate complement or ADCC.
Anti-idiotype specificity may be used. Specificities for widely found idiotypes such as that which may be provided by the commonly used DP-47 V gene germline sequence may be used to recruit any antibody where that idiotype is still recognisable in the mature antibody. Specificity for idiotypes of specific antibodies is useful for using the antibody displaying that idiotype in an agglutination assay. To use diabodies as an example, a diabody molecule directed against a cell surface marker and the idiotype of the antibody would bridge one cell to the antibody. A second diabody molecule would be able to bind to another antigen binding site on the antibody and to a second cell thus crosslinking them. IgM molecules would be particularly suitable for this, because they have 10 antigen binding sites per molecule.
The multi-specific binding substance may be bi-specific. It may be a bi-specific antibody or antibody fragment (as discussed). Preferably, it is a xe2x80x9cdiabodyxe2x80x9d, ie a multimer (e.g. dimer) of polypeptides each of which have a first domain comprising a binding region of an immunoglobulin heavy chain variable region and a second domain comprising a binding region of an immunoglobulin light chain variable region, the two domains being linked but not able to associate to form an antigen binding site. The linkage may be by a peptide linker of xe2x88x921 to about 10 amino acids (e.g. 5). The polypeptides associate into multimers wherein the first domain of one polypeptide associates with the second domain of another polypeptide to form an antigen binding site. For further information and possibilities of formats for a xe2x80x9cdiabodyxe2x80x9d for use in the present invention, refer to WO 94/13804. Also preferred are scFv dimers, wherein each polypeptide comprises heavy and light chain variable region binding regions which can associate intra-molecularly to form antigen binding site (in contrast to diabodies) because the peptide limber joining the two domains in each polypeptide is long enough, and (Fab)2.
A method according to the present invention may be carried out in vitro or in vivo where it may be a method of treatment of an individual for a condition wherein recruitment of antibody mediated effector function is, or is likely to be, of benefit. Administration to an individual may be using any standard technique, the criteria for selection of a technique and selection of dosages, frequency of administration etc, being well known to those skilled in the art. Administration of antibody is described, for example, in Hale et al, Lancet, ii, 1394-1399 (1988), Simmons et al, Circulation, 89, 596-603 (1994) and Riethmuller et al, Lancet, 343, 1177-1183 (1994).
In vitro, use may be made of a multispecific binding substance, such as a bispecific antibody such as a diabody, in retargetting antibodies to recruit antibody effector function to treat target cells/tissue removed from a patient. For instance, bone marrow from a patient with leukaemia may be taken and the cells treated, ex vivo, with a binding substance such as a bispecific diabody directed against a marker specific for the tumour cells and an immunoglobulin IgG1 constant region, together with IgG1 antibody and complement. Tumour cells would then be specifically lysed and the whole cells remaining may be taken and returned to the patient. Alternatively, ADCC may be used, the binding substance (e.g. diabody) together with IgG1 and a preparation of killer cells being added to the bone marrow cells to lyse the tumour cells before returning the remaining cells to the patient.
Similarly, e.g. using complement lysis, the recruitment of effector function may be used in a diagnostic assay for the number of cells expessing a particular marker, e.g. tumour specific antigen, present in a sample e.g. of blood. The degree of lysis would reflect the number of cells present. If an anti-IgM binding substance (e.g. diabody) plus IgM were used, the increased complement lysis would increase the sensitivity to detect very small numbers of tumour cells expressing cell surface markers.
Mediation of effector function may be caused or allowed according to conditions under which the invention is operated. For instance, in vitro mediation may be caused by addition into the medium of required components of the effector system (e.g. complement). However, in serum, for example, either in vitro or in vivo all necessary components for effector function may be present ab initio, allowing effector function to be called down upon binding of the multi-specific binding substance to the target and to antibody.
A further aspect of the invention provides the use of a multi-specific binding substance in the recruitment of an antibody-mediated effector function to a target, the binding substance having an anti-antibody binding specificity and binding specificity for the target. The use may be made of the multi-specific binding substance in any method provided by the present invention. Use may be in the manufacture of a medicament for recruitment of antibody mediated effector function, e.g. for the treatment of a condition wherein this is, or is likely to be, of benefit (see above).
Pharmaceutical compositions comprising multi-specific binding substances as disclosed, and use of such compositions, are also provided by the present invention. Such pharmaceutical compositions may comprise any suitable pharmaceutically acceptable excipient.
Another aspect of the present invention provides a multi-specific (e.g. bispecific) binding substance e.g. xe2x80x9cdiabodyxe2x80x9d (as disclosed) having an anti-antibody binding specificity (and a binding specificity for a target). Such a multi-specific binding substance has a binding site with anti-antibody binding specificity and a binding site with binding specificity for a target, and comprises a multimer of polypeptides, each polypeptide having a first domain comprising a binding region of an immunoglobulin heavy chain variable region and a second domain comprising a binding region of an immunoglobulin light chain variable region, the binding sites being formed by association of a first domain of one polypeptide in the multimer with a second domain of another polypeptide in the multimer. In a diabody, the first domain of each polypeptide is unable to associate with the second domain of that polypeptide to form an antigen binding site. Compositions comprising such a multimer, e.g. pharmaceutical compositions which may include a pharmaceutically acceptable excipient, are also provided by the invention. The diabody may be a polypeptide dimer.
In addition to utility in the methods and compositions disclosed supra, such a multispecific binding substance finds utility in a further aspect of the present invention, namely, a general method of targeting or recruiting an antibody to a target for which the antibody has no binding specificity, either with or without associated effector function. For instance a multi-specific (e.g. bispecific) diabody may be used in agglutination assays.
Multispecific binding substances such as the preferred diabodies (e.g. bispecific) may be used for coagulation of cells, bacteria or viruses, by making multiple interactions, as with diagnostic assays of agglutination of red blood cells, to determine for instance, blood cell types. Diabodies with one arm directed against an antibody molecule may be used in different formats to link together cells, as illustrated in FIG. 2.
For example, a diabody or other multi-specific binding substance may be used which has one arm directed against a cell surface antigen and another directed against IgM. The multivalent nature of IgM means that two or more diabody molecules may bind to the IgM molecule and thus crosslink between different blood cells. This IgM may be added as an extra reagent or it may be possible to use the IgM present in blood samples tested to promote the agglutination.
One arm may be directed against a cell surface antigen and the another directed against an idiotype commonly found in antibody molecules, such as antibodies directed against elements of the DP-47 VH gene, a gene segment commonly used in human antibodies (Tomlinson et al, J. Mol. Biol. 227 776-798 (1992)). IgM molecules with this idiotype would be particularly useful.
One arm may be directed against isotypes other than IgM for use in agglutination assays, but since these other antibodies are smaller, they may be less effective in agglutinating cells.
In any embodiment of the present invention the target may be any antigen e.g. of bacterial, viral, fungal, protozoal origin or antigen on the surface of cells (e.g. cancer cells), enabling recruitment of the natural antibody encoded effector functions to the targets displaying those antigens (e.g. bacteria, viruses, parasites or tumor cells) by way of a multi-specific binding substance which has binding specificity for the antigen and anti-antibody binding specificity.
Further aspects of the invention will be apparent to persons skilled in the art.
The following examples illustrate how the principles disclosed herein may be put into practice. Those skilled in the art will readily appreciate modifications and variations which may be made without departing from the invention disclosed herein.
All documents mentioned in the text are herein incorporated by reference.